The present invention relates generally to assay analyzing systems and, more particularly, concerns a system and method for creating digital images of randomly arranged specimens (e.g. beads within gels, colonies within petri dishes) or specimens arranged in regular arrays (e.g. wells in plastic plates, dots spotted onto membranes). The invention is capable of creating digital images and performing automated analyses of specimens which emit very low levels of fluorescence, chemiluminescence, or bioluminescence. More particularly, the invention is designed for the analysis of luminance arising from assays within well plates and gel media, and on membranes, glass, microfabricated devices, or other supports.
Many chemical and molecular biological assays are designed so that changes in the absorbance, transmission, or emission of light reflect reactions within the specimen. Therefore, instruments used to quantify these assays must detect alterations in luminance.
Wells. Some assays are conducted within discrete flasks or vials, while others are performed within plastic plates fabricated to contain a number of regularly spaced wells. xe2x80x9cWell platexe2x80x9d assays are higher in throughput and lower in cost than similar assays in discrete containers. Standard well plates contain 96 wells in an area of 8xc3x9712 cm. The trend is to higher numbers of wells, within the same plate size. Today""s highest commercial density is 384 wells. Very high density arrays of small wells (microwells, e.g. thousands/plate with a fill volume of less than 1 ul/well) are under development, and will become commercially available as microwell filling and detection technologies mature.
Dot blots. Grids of small dots (reactive sites) are placed onto flat support membranes or slips of treated glass. A high density grid can contain many thousands of discrete dots. Grid assays usually involve hybridization with synthetic oligonucleotides, to look for genes containing specific sequences, or to determine the degree to which a particular gene is active. Applications include library screening, sequencing by hybridization, diagnosis by hybridization, and studies of gene expression. High density grids provide the potential for very high throughput at low cost, if analyzing the grids can be made simple and reliable. Therefore, considerable commercial attention is directed at companies developing technology for creating, detecting, and analyzing high density arrays of genomic sequences.
Combinatorial assays. Some assays involve small particles (typically beads coated with compounds) which act as the reactive sites. There might be many thousands of beads, each coated with a different compound (e.g. molecular variants of an enzyme) from a combinatorial library. These beads are exposed to a substance of interest (e.g. a cloned receptor) in wells, or in a gel matrix. The beads which interact with the target substance are identified by fluorescence emission or absorption in the region around each bead. Beads which interact are surrounded by faint areas of altered luminance. Very sensitive detectors are required to identify the subtle alterations in luminance around the beads that interact with the target.
Electrophoretic separations. A solubilized sample is applied to a matrix, and an electrical potential is applied across the matrix. Because proteins or nucleic acids with different amino acid or nucleotide sequences each have a characteristic electrostatic charge and molecular size, components within the sample are separated by differences in the movement velocities with which they respond to the potential. The separated components are visualized using isotopic, fluorescent, or luminescent labels. In many cases (e.g. chemiluminescence), the luminance from the specimen is very dim.
Assays which occur within a regularly spaced array of active sites (wells, dot blots within a grid) can be referred to as fixed format assays. Assays which involve specimens that are irregularly distributed within a gel or blot matrix can be termed free format assays.
Fixed format assays are usually performed without imaging. In contrast, free format assays require the use of image analysis systems which can detect and quantify reactions at any position within an image.
Instruments designed for fixed format assays generally lack imaging capabilities, and have not been applied to free formats. Similarly, very few imaging instruments designed for free formats have been applied to wells, and other fixed format targets.
Nonimaging counting systems (liquid scintillation counters, luminometers, fluorescence polarization instruments, etc.) are essentially light meters. They use photomultipliers (PMTs) or light sensing diodes to detect alterations in the transmission or emission of light within wells. Like a light meter, these systems integrate the light output from each well into a single data point. They provide no information about spatial variations within the well, nor do they allow for variation in the packing density or positioning of active sites.
Each PMT reads one well at a time, and only a limited number of PMTs can be built into a counting system (12 is the maximum in existing counting systems). Though the limited number of PMTs means that a only few wells are read at a time, an array of wells can be analyzed by moving the PMT detector assembly many times.
The major advantages of nonimaging counting systems are that they are a xe2x80x9cpush-buttonxe2x80x9d technology (easy to use), and that the technology is mature. Therefore, many such instruments are commercially available, and their performance is well-characterized.
The major disadvantages of counting systems are:
a. Limited flexibilityxe2x80x94few instruments can cope with 384 wells, and higher density arrays of fluorescent or luminescent specimens are out of the question.
b. Fixed format onlyxe2x80x94designed as well or vial readers, and cannot read specimens in free format.
c. Slow with dim assaysxe2x80x94although scanning a few wells at a time can be very fast when light is plentiful, dim assays require longer counting times at each position within the scan. As there are many positions to be scanned, this can decrease throughput.
In summary, non-imaging counting systems are inflexible and offer limited throughput with some specimens.
Scanning Imagers
For flat specimens, an alternative to nonimaging counting is a scanning imager. Scanning imagers, such as the Molecular Dynamics (MD) Storm, MD FluorImager, or Hitachi FMBIO pass a laser or other light beam over the specimen, to excite fluorescence or reflectance in a point-by-point or line-by-line fashion. Confocal optics can be used to minimize out of focus fluorescence (e.g. the Biomedical Photometrics MACROscope), at a sacrifice in speed and sensitivity. With all of these devices, an image is constructed over time by accumulating the points or lines in serial fashion.
Scanning imagers are usually applied to gels and blots, where they offer convenient operation. A specimen is inserted and, with minimal user interaction (there is no focusing, adjusting of illumination, etc.), the scan proceeds and an image is available. Like the nonimaging counting system, the scanning imager is usually a push-button technology. This ease of use and reasonably good performance has lead to an increasing acceptance of scanning imagers in gel and blot analyses.
Scanning imagers have four major shortcomings:
a. Slow scanning. The beam and detector assembly must be passed over the entire specimen, reading data at each point in the scan. Scanning a small specimen could easily take 5-10 minutes. A large specimen might take xc2xd hour to scan. This slow scan limits throughput, and complicates the quantification of assays that change during the scan process.
b. Limited number of wavelengths. A limited number of fluorescence excitation wavelengths is provided by the optics. Therefore, only a limited number of assay methods can be used.
c. Low sensitivity. Most scanning imagers exhibit lower sensitivity than a state of the art area imager.
d. Not appropriate for luminescence. Scanning imagers require a bright signal, resulting from the application of a beam of light to the specimen. Therefore, specimens emitting dim endogenous luminescence (e.g. reactions involving luciferase or luminol) cannot be imaged.
e. Not appropriate for wells. Only flat specimens can be imaged. A limited number of confocal instruments can perform optical sectioning and then reconstruct the sections into a focused thick image.
An area imaging system places the entire specimen onto a detector plane at one time. There is no need to move PMTs or to scan a laser, because the camera images the entire specimen onto many small detector elements (usually CCDs), in parallel. The parallel acquisition phase is followed by a reading out of the entire image from the detector. Readout is a serial process, but is relatively fast, with rates ranging from thousands to millions of pixels/second.
Area imaging systems offer some very attractive potential advantages.
a. Because the entire specimen is imaged at once, the detection process can be very quick.
b. Given an appropriate illumination system, any excitation wavelength can be applied.
c. Luminescence reactions (emitting light without incident illumination) can be imaged, including both flash and glow bioluminescence or chemiluminescence.
d. Free or fixed format specimens can be imaged.
Luminescence imaging is more easily implemented, in that illumination does not have to be applied. However, most luminescence reactions are quite dim, and this can make extreme demands upon existing area imaging technology. The standard strategy is to use sensitive, cooled scientific grade CCD cameras for these types of specimens. However, in the absence of the present invention, integrating cameras will fail to image many luminescent specimens. Therefore, the present invention can image specimens that other systems cannot.
Typical prior art systems apply area imaging to luminescent assays on flat membranes and luminescent assays in wells. Standard camera lenses are always used. The results of well imaging are flawed, in that there is no correction for parallax error.
There is more extensive prior art regarding use of area imaging in fluorescence. Fluorescence microscopy (see Brooker et al. U.S. Pat. No. 5,332,905) and routine gel/blot imaging are the most common applications. Prior art in microscopy has little relevance, as no provision is made for imaging large specimen areas.
The existing art relating to macro specimens is dominated by low cost commercial systems for routine gel/blot fluorescence. These systems can image large, bright areas using standard integrating CCD cameras. However, they have major disadvantages:
a. Limited to the wavelengths emitted by gas discharge lamps. Typically some combination of UVA, UVB, UVC, and/or white light lamps is provided. Other wavelengths cannot be obtained.
b. Wavelengths cannot be altered during an assay. If the illumination must be changed during the assay (e.g. as for calcium measurement with fura-2), the devices cannot be adapted.
c. Insensitive to small alterations in fluorescence. Transillumination comes from directly below the specimen into the detector optics. Therefore, even very good filters fail to remove all of the direct illumination, and this creates a high background of nonspecific illumination. Small alterations in fluorescence (typical of many assays) are lost within the nonspecific background.
d. Inefficient cameras and lenses. A very few systems use high-performance cameras. Even these few systems use standard CCTV or photographic lenses, which limit their application to bright specimens.
e. Parallax error precludes accurate well imaging. As fast, telecentric lenses have not been available, these systems exhibit parallax error when imaging wells.
Novel features of the present invention minimize the disadvantages of known macro fluorescence systems. These novel features include:
a. Illumination wavelengths may be selected without regard to the peak(s) of a gas discharge lamp or laser.
b. Using a computer-controlled filter wheel or other device, illumination may be altered during an assay,
c. Small alterations in fluorescence emission can be detected. Because fluorescence illumination comes via epi-illumination, or from a dorsal or lateral source, direct excitation illumination does not enter the optics. This renders the nonspecific background as low as possible.
d. Very efficient camera and lens system allow use with dim specimens.
e. Unique telecentric lens is both very fast, and removes parallax error so well plate assays are accurate.
A primary advantage of the present invention is its fast, telecentric lens, which can image an entire well plate at once, and which can provide efficient epi-illumination to transparent or opaque specimens. Fiber optic coupling to the specimen can be used instead of lens coupling. For example, a fiber optic lens has been used with an image intensified CCD camera run in photon counting mode for analyses of data in fixed or free formats. This approach yields good sensitivity, but has the following major disadvantages:
a. Although it is suggested that the system could be used with fluorescent specimens, it would be limited to specimens that are transilluminated, because there is no place to insert an epi-illumination mechanism. Therefore, the fiber lens system would have degraded sensitivity, and could not be used with opaque specimens. Many specimens are opaque (e.g. many well plates, nylon membranes).
b. Well plates are 8xc3x9712 cm. Image forming fiber optics of this size are very difficult and expensive to construct. Therefore, the specimen would have to be acquired as a number of small images, which would then be reassembled to show the entire specimen.
This multiple acquisition would preclude use of the device with assays which change over time.
An area imaging analysis system (LUANA) is disclosed by D. Neri et al. (xe2x80x9cMultipurpose High Sensitivity Luminescence Analyzerxe2x80x9d, Biotechniques 20:708-713, 1996), which uses a cooled CCD, side-mounted fiber optic illuminator, and an excitation filter wheel to achieve some functions similar to the present invention (selection of wavelengths, area imaging). However, LUANA uses a side-mounted fiber optic, which is widely used in laboratory-built systems, and creates problems that are overcome by the present invention. Specifically, use of a side-mounted fiber optic provides very uneven illumination, particularly when used with wells. The epi- and transillumination systems of the present invention provide even illumination of both flat specimens and wells. Further, in LUANA, parallax would preclude imaging of assays in wells.
Another system (Fluorescence Imaging Plate Readerxe2x80x94FLIPR of NovelTech Inc., Ann Arbor Mich.) uses an area CCD to detect fluorescence within 96 well plates. This device is a nonimaging counting system, and uses the area CCD instead of multiple PMTs. To achieve reasonable sensitivity, it runs in 96 well format and bins all pixels within each well into a single value. The device is not applicable to luminescence imaging, free format imaging, or higher density well formulations and is very costly.
There is extensive prior art in the use of imaging to detect assays incorporated within microfabricated devices (e.g. xe2x80x9cgenosensorsxe2x80x9d). Some genosensors use scanning imagers, and detect emitted light with a scanning photomultiplier. Others use area CCDs to detect alterations at assay sites fabricated directly onto the CCD, or onto a coverslip that can be placed on the CCD. Genosensors have great potential when fixed targets are defined. For example, a chip is fabricated that looks for a specific sequence of genomic information, and this chip is used to screen large numbers of blood samples. While highly efficient for its designed sequence, the chip has to contain a great number of active sites if it is to be useful for screening a variety of sequences. Fabrication of chips with many thousands of sites is costly and difficult. Therefore, the first generation of genosensors will be applied to screening for very specific sequences of nucleotides.
The inflexibility of the microfabricated device contrasts with the present invention, which does not require microfabrication of the assay substrate. Instead, the present invention permits assays to be conducted in wells, membranes, silicalized slides, or other environments. Almost any reaction may be quantified. Thus, the present invention could be used as an alternative technology to microfabrication. Because the present invention is flexible, and allows almost any chemistry to be assayed, it can be used for all phases of assay development. These include prototyping, and mass screening. The invention therefore provides an alternative to microfabrication, when microfabrication is not feasible or cost-effective.
Each of the prior art references discussed above treats some aspect of imaging assays. However, the prior art does not address all of the major problems in imaging large specimens at low light levels. The major problems in low light, macro imaging are:
a. very high detector sensitivity required;
b. flexible, monochromatic illumination of large areas is required;
c. parallax error must be avoided; and
d. more reliable procedures are needed to find and quantify targets.
Broadly, it is an object of the present invention to provide an imaging system for assays which overcomes the shortcomings of prior art systems. It is specifically intended to provide a complete system for the area imaging of assays in wells and on membranes. It is specifically contemplated that the invention provide a complete system for the area imaging of chemiluminescent, fluorescent, chemifluorescent, bioluminescent, or other nonisotopic hybridization assays, including high density dot blot arrays.
It is another object of the invention to image chemiluminescent, fluorescent, chemifluorescent, bioluminescent, or other nonisotopic assays, including combinatorial assays, in free format.
It is an object of the invention to provide software for digital deconvolution of the fluorescence image data. Application of the software decreases flare and out of focus information.
It is also an object of the present invention to provide a method and system for imaging assays which are flexible, reliable and efficient in use, particularly with low level emissions.
The present invention provides synergistic combination of detector, lens, imaging system, and illumination technologies which makes it able to image the types of specimens previously acquired with nonimaging counters and scanning imagers. In particular, it can be used with fixed or free formats, and with wells or flat specimens. It is able to detect fluorescence, luminescence, or transmission of light.
The features of the invention include that it detects and quantifies large arrays of regularly spaced targets, that it detects and quantifies targets that are not arranged in regular arrays, and that it performs automated analyses of any number of regularly spaced specimens, from small numbers of large wells to large numbers of very small wells or dot blots.
It is another feature of the invention to provide an area illumination system that: can deliver homogenous monochromatic excitation to an entire well plate or similarly sized specimen, using standard and low cost interference filters to select the excitation wavelength; and can deliver varying wavelengths of homogenous monochromatic excitation to an entire well plate or similarly sized specimen, under computer control.
A system embodying the invention provides a lens designed specifically for assays in the well plate format. This lens is very efficient at transferring photons from the specimen to the CCD array (is fast), preferably contains an epi-illumination system, and can be used with very dim specimens. The lens is also telecentric. A telecentric lens has the property that it peers directly into all points within a well plate, and does not exhibit the parallax error that is characteristic of standard lenses.
A preferred system provides a telecentric and fast lens that generates an even field of epi-illumination, when required. The lens is equipped with an internal fiber optic illumination system, that does not require a dichroic mirror. Preferably, the lens is constructed to accept an internal interference filter used as a barrier filter. Light rays passing through the lens are almost parallel when they strike the barrier filter, so that the filter operates at its specified wavelength and bandwidth tolerance.
It is a feature of the invention that it provides high light gathering efficiency, whether used with a fast telecenric lens, or standard photographic lenses.
A preferred system provides a CCD area array camera that has high quantum efficiency (approximately 80%), and high-sensitivity (16 bit precision), so that most specimens can be detected by integration without intensification. Preferably, the system has an integrating, cooled CCD camera which has coupled thereto an optional image intensifier. In an embodiment intended for extremely low light levels, incident illumination from the specimen is amplified by the intensifier, and the amplified light is accumulated onto the integrating camera over an integration period. At the end of the integration period, the camera is read out to a dedicated controller or imaging apparatus to reproduce the light image. Multiple exposures may be used to increase the dynamic range of the camera. A light-tight specimen chamber is provided, to which all illumination and detection components may be mounted, and which contains the specimens.
A system in accordance with the invention may incorporate a translation stage (optional), that may be housed within the light-tight chamber and used to move large specimens (e.g. 22xc3x9722 cm membranes) past the optical system. The invention controls the stage motion through software, and that creates a single composite image from the multiple xe2x80x9ctilesxe2x80x9d acquired with the translation stage.
Preferably, the invention provides software control that corrects the shading, geometric distortion, defocus, and noise errors inherent to the camera and lens system; and that removes as much nonspecific fluorescence as possible, using multiple images created with different excitation filters.
In particular, the invention provides software to deconvolve images from a single focal plane, using optical characteristics previously measured from the lens and detector system. It should be appreciated that data from multiple focal planes may also be deconvolved.
While the preferred embodiment of the invention uses a high-precision, cooled CCD camera, if cost is a major factor, the present invention could be constructed using lower cost integrating cameras. In this case, shorter integration periods can be achieved, with a reduction in image quality and ultimate sensitivity.